The desirability of having an immunoassay capable of being employed by hospitals and clinical laboratories for the early detection of viral infections has been widely recognized. The need for such an assay can be illustrated, for example, by acute hepatitis B, one form of the various hepatitis viral infections. Acute hepatitis B causes significant mortality and morbidity and chronic infection with this virus is also associated with hepatocellular carcinoma, chronic active hepatitis and cirrhosis. In view of this need, much research has been devoted to the development of immunoassays capable of detecting viral antigens such as hepatitis B surface antigen (HBsAg), the viral marker for hepatitis B, in the blood stream of patients.
Generally, the immunoassays which have been developed to date can be classified as radioimmunoassays (RIA) or immunoradiometric assays (IRMA). In a RIA, the amount of antigen present in a sample is measured indirectly employing a limited amount of antibody to compete for labeled antigen. In an IRMA, antigen is assayed directly by reacting the antigen with excess labeled antibody.
In one class of IRMA assays, the unknown antigen is insolubilized and reacted with labeled antibody. When the antigen is insolubilized by reaction with solid-phase antibody, the assay is termed a "two-site IRMA", "junction test", or "sandwich assay". Sandwich assays are further classified according to their methodology as forward, reverse or simultaneous sandwich assays.
Heretofore, the most widely used immunoassay for viral hepatitis B has been the forward sandwich assay. In this assay, a patient sample containing HBsAg is initially incubated with a solid-phase immunoadsorbent containing immobilized antibody for HBsAg. Incubation is continued for a sufficient period of time to allow HBsAg in the patient sample to bind to immobilized antibody on the solid-phase immunoadsorbent. After this first incubation, the solid-phase immunoadsorbent is separated from the incubation mixture and washed to remove excess antigen and other interfering substances, such as non-specific binding proteins, which also may be present in the patient sample. The solid-phase immunoadsorbent containing HBsAg bound to immobilized antibody is subsequently incubated for a second time with labeled antibody capable of binding to HBsAg, which is multivalent. After the second incubation, another wash is performed to remove unbound labeled antibody from the solid-phase immunoadsorbent thereby removing non-specifically bound labeled antibody. Labeled antibody bound to the solid phase immunoadsorbent is then detected and the amount of labeled antibody detected serves as a direct measure of the amount of HBsAg present in the original patient sample. Such forward sandwich assays are described in the patent literature, and in particular, in U.S. Pat. Nos. 3,867,517 and 4,012,294, issued to Chung-Mei Ling.
Despite their widespread use, forward sandwich assays do have certain inherent disadvantages. For example, the requirement to perform two separate incubations as well as two separate washings means that the assay is relatively time-consuming and requires a number of manipulative operations which must be performed by skilled technicians thereby making the forward sandwich assay relatively costly. In addition, the kinetics in the second incubation are relatively slow since labeled antibody must react in this step with antigen bound to antibody immobilized on the solid-phase immunoadsorbent.
The reverse sandwich immunoassay was developed to overcome some of the disadvantages of the forward sandwich assay. In a reverse sandwich assay, the patient sample is initially incubated with labeled antibody after which the solid-phase immunoadsorbent containing immobilized antibody is added and a second incubation is carried out. Thus, the initial washing step of a forward sandwich assay is not required, although a wash is performed after the second incubation period. The major advantages to the reverse sandwich assay compared to the forward sandwich assay are the elimination of the first wash step and the improvement in kinetics of reaction since the labeled antibody and antigen in the patient serum are initially incubated together. Thus, for a total incubation period less than that required to reach equilibrium, it would be expected that the sensitivity of the reverse assay would be higher than that of the forward assay. A reverse sandwich assay has been described in the patent literature in U.S. Pat. No. 4,098,876, issued to Roger N. Piasio et al.
Although reverse sandwich assays have potential advantages over forward sandwich assays, they still require two separate incubation periods and one washing.
Simultaneous sandwich assays, although known, have not found widespread use. In a simultaneous sandwich assay, the patient sample, immunoadsorbent having immobilized antibody thereon, and labeled antibody are incubated simultaneously in one incubation step. Although offering certain potential advantages due to the single incubation and lack of washing steps required, simultaneous sandwich assays have previously been found to be less sensitive and reliable than forward and reverse sandwich assays. This would be expected, of course, since the patient sample often contains many non-specific binding molecules, such as proteins, which are not washed from the assay as they are in the forward sandwich assay.